Our main project deals with understanding the organization of the unique grid-like network of skeletal muscle fiber microtubules and its implication in muscle diseases, particularly Duchenne muscular dystrophy (DMD). Results obtained in previous years by Sarah Oddoux, Kristien Zaal and others using fluorescent protein constructs expressed in muscle fibers ex vivo and in vivo showed that this microtubule network, apparently static, is in fact composed of highly dynamic microtubules moving along each other as along tracks (Oddoux et al., 2013). We now work on understanding how microtubule organization in the mdx mouse, the mouse model for DMD, differs from that in wild-type (WT) mice. Mdx muscle fibers have an abnormal microtubule network; instead of a regular grid, microtubules are disordered, dense, and mostly oblique. Software developed in the Light Imaging Section for the analysis of microtubule directionality is essential in the automated assessment of muscle microtubules (Liu et al., 2014). Microscopy on live fibers has been carried out to compare mdx and WT muscle microtubule dynamics. We hypothesized that mdx microtubules would mostly differ by their orientation/directionality, not by their growth characteristics. The results obtained, so far, confirm our predictions. Furthermore, the results demonstrate that the differences in microtubule orientation can be observed as soon as microtubules start growing from the nucleating Golgi elements. Thus muscle microtubules grow as if Golgi elements themselves, or the nucleating molecules anchored to the Golgi elements, had a specific orientation, disturbed in mdx muscles. Super-resolution microscopy will be used to investigate the orientation of the Golgi elements. A secondary project has investigated the CLARITY technique for making whole muscle transparent in fluorescence microscopy. The initial technique (Chung & Deisseroth, Nat. Meth. 2013, 10 (6) 508-13) has been applied to the brain. The goal is to make the tissue transparent to allow the analysis of large structures such as nerve tracts in the brain. Lipid membranes are removed by a process that embeds the tissue in a gel, while simultaneously fixing proteinaceous structures. We thought that it would be advantageous to apply this technique to skeletal muscle, in order to follow nerve and vascular organization, muscle fiber distribution, and their changes in muscle diseases. Andy Milgroom succeeded in importing the technique to LIS and obtained the first example of CLARITIzed skeletal muscle. Manuscripts are currently prepared from both projects.